Process for preparing an autologous platelet gel and membrane thereof

ABSTRACT

A process is disclosed for preparing an autologous platelet gel and membranes thereof comprising mixing a platelet concentrate with a calcium salt and batroxobin. This process encompassing the use of batroxobin as the gel activator allows to overcome the prior art processes drawbacks connected with the use for the same purpose of human of bovine thrombin. A kit is also described for carrying out this process.

FIELD OF THE INVENTION

The present invention relates to a process for preparing an autologousplatelet gel and membranes thereof, and a kit for carrying out thisprocess.

BACKGROUND OF THE INVENTION

Marx and colleagues have shown that the use of platelet concentraterepresents an innovative method to modulate and accelerate wound healingprocess, as well as bone regenerative processes (Oral surg., Oral Med.,Oral Path 1998, 85: 638-646). Therefore these authors elaborated atechnique allowing to isolate and precipitate platelets therebyobtaining an autologous platelet concentrate, which is neither toxic norimmunoreactive. This substance is able to enhance the effects of growthfactors contained in platelets granules and which are initiators ofmetabolic pathway involved in wound healing steps.

According to Marx's protocol encompassing the use of a cellularseparator, the platelet concentrate is prepared by carrying out a venousblood sample of 500 ml, namely an operation which is accomplished bymeans a venous catheter. The platelet concentrate is subsequentlyactivated with calcium chloride and bovine thrombin to produce aplatelet gel which can be combined with either autologous spongy andcortical bone or bone matrix consisting of bio-glasses.

In Italy Marx technique for the activation of a platelet gel cannot becarried out since bovine thrombin is no longer available on the market.In addition this method involving the use of very high amounts of bloodrequiring specific instrumentation and skilled personnel for preparingplatelet concentrate, cannot be realised in a medical surgery so as toallow a routine use of the platelet gel.

In U.S. Pat. No. 5,585,007 and WO 98/11925 methods for preparingplatelet gel are disclosed. These methods, although representing animprovement if compared to Marx's protocol, as they necessitate muchlower blood amounts (50-60 ml), they require for platelet gel formationthe use of bovine or human thrombin.

To date human thrombin can only be prepared by DNA recombinanttechnique. In Italy substances prepared according to this technique canbe used only for in vitro experiments, but not for in vivo experiments.

Technical Problem

The need was felt of a process for preparing a platelet gel notpresenting the prior art processes drawbacks, connected with the use asthe activator of human or bovine thrombin.

SUMMARY OF THE INVENTION

The Applicant has unexpectedly found that it is possible to overcome theaforementioned drawback, by using as the activator batroxobin in placeof thrombin.

The present invention therefore relates to a process for preparing aplatelet gel comprising mixing a platelet concentrate with an inorganicor organic calcium salt and batroxobin.

A further object of the present invention relates to a kit for carryingout the process according to the present invention which in particularcomprises:

-   -   a) a monouse sterile capsule of transparent plastic or glass        material, fitted with a screw cap constituted in the upper part        by a perforable membrane coated with a suitable sterile coating,        said perforable membrane allowing the insertion of the needle of        a syringe containing the platelet concentrate, and        contemporaneously avoiding the air contact with the inside of        the capsule,    -   b) two plug injectors surmounting said perforable membrane        containing respectively the calcium salt and batroxobin.

Finally further objects of the present invention are:

-   -   the autologous platelet gel obtained with the process according        to the present invention.    -   a membrane comprising the autologous platelet gel according to        the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 and 2 represent two preferred embodiments of the kit accordingto the present invention.

In particular in these figures: (3) represents the sterile monousecapsule, (4) the screw cap constituted in the upper part by a perforablemembrane (5). (1) and (2) represent the two plug injectors surmountingsaid perforable membrane (5). Finally (6) represents the syringecontaining the platelet concentrate, and (7) platelet gel formed duringthe reaction.

DETAILED DESCRIPTION OF THE INVENTION

1. Preferred Operating Conditions of the Process According to thePresent Invention

Preferably the inorganic calcium salt used in the process according tothe present invention is calcium chloride, whereas when an organiccalcium salt is sued this is preferably calcium gluconate. According toa particularly preferred embodiment, the process is carried out by usingcalcium gluconate.

The inorganic or organic calcium salt is in the form of an aqueoussolution containing it in amounts ranging from 5.10⁻⁵ to 0.5 M.

According to a preferred embodiment in the process of the presentinvention, 8.10⁻⁵ M (80 μM) calcium chloride is utilised.

A more preferred embodiment foresees the use of 0,23 M calciumgluconate. The batroxobin used is preferably in the form of an aqueoussolution having 1 thrombin international unit and is sold under thecommercial name Botropase by Ravizza Farmaceutici S.p.A.

2. Method for Preparing the Platelet Concentrate

The platelet concentrate used in the process according to the presentinvention is prepared from whole blood (sample of 40-50 ml of venousblood), collected in 20 ml syringes, containing 4 ml sodium citrate usedas anticoagulant ), by a two phases centrifugation, thereby obtaining anintermediate product being platelet rich plasma. This method allows torecover at least 80% of platelets present in whole blood.

In particular this technique encompasses the following steps. Venousblood is collected in 4 tubes and centrifuged at 180 g for 20 minutes.After this treatment two phases are obtained a dark one constituted byprecipitated red and white cells at the bottom of the tubes, and a clearone visible in the upper part of the probe, consisting of platelet richplasma. This plasma is taken and transferred by means of a Pasteurpipette into other 4 tubes and centrifuged at 580 g for 20 minutes.Thanks to this faster centrifugation (580 g versus 180 g) it is possibleto obtain platelets sedimentation as a dense small bottom said “pellet”.The liquid supernatant consists of platelet poor plasma.

The tubes are emptied, however leaving in each of them a small aliquot(1.5 ml) of platelet poor plasma necessary to suspend once again the“pellet” thereby obtaining a homogeneous suspension.

Thus proceeding it is possible to obtain a final platelet concentrate(of about 6 ml) deriving from the unification of the plateletssuspensions present in the 4 tubes. This platelet concentrate after arest period of about 15 minutes at room temperature, is ready to be usedas reactant in the process according to the present invention.

3. Membranes According to the Present Invention and Method for PreparingThem

The membrane according to the present invention may contain activeprinciples such as antibacterial agents, disinfectants, and/or sterileexcipients etc.

Preferably the membrane according to the present invention essentiallyconsists of the autologous platelet gel according to the presentinvention.

These membranes are obtained by simply eliminating the liquid in excess.

For example if the platelet gel preparation occurs in plastic or glassPetri dishes (like those utilised in cellular cultures), it is possibleto obtain a thin membrane, as the gel during the activation takes theform of the container.

In this case by eliminating the liquid in excess by means for example ofa Pasteur pipette released during the gel formation it is possible toobtain a pliable membrane having a well defined contour.

The removed liquid in excess consisting of batroxobin and calcium saltis added to the platelet poor plasma coming from the secondcentrifugation and determines after 20-30 minutes the formation of afurther thick platelet gel.

4. The Kit According to the Present Invention

FIGS. 1 and 2 represent preferred embodiment of the kit according to thepresent invention.

In particular in these figures: (3) represents the sterile monousecapsule, (4) the screw cap constituted in the upper part by a perforablemembrane (5). This membrane is coated with a sterile coating normallyused in phamaceutical and diagnositc industry such as aluminum film oranother equivalent material. (1) and (2) represent the two pluginjectors surmounting said perforable membrane (5) and containingrespectively the inorganic or organic calcium salt (more preferably 0.23M calcium gluconate), and batroxobin. Finally (6) represents the syringecontaining the platelet concentrate, and (7) platelet gel formed duringthe reaction. A manual pressure onto the cap injector (1) allows theoutlet of calcium gluconate in amounts of from 0.3 to 0.5 ml into thecapsule (3). Analogously a manual pressure onto the cap injector (2)allows the outlet of batroxobin in amounts of from 0.3 to 0.5 ml. Thecapsule (3) reported in FIG. 1 has preferably a capacity of from 10 to20 ml, whereas the same capsule of the kit reported in FIG. 2 permits toobtain membranes of diameter of from 3 to 9 cm as the capsule may have adiameter ranging from 3.5 to 10.0 cm.

The process according to the present invention carried out with theaforementioned kit encompasses the following steps:

-   -   A) charging a 10 ml syringe (6) with a platelet concentrate        amount ranging from 3 to 9 ml;    -   B) removing the sterile coating from the perforable membrane (5)        and introducing the needle of the syringe into the capsule (3)        through the perforable membrane (5);    -   C) pressing in rapid succession the cap injectors (1) and (2)        containing respectively the calcium gluconate and batroxobin and        the piston of the syringe (6) inside the capsule (3);    -   D) gently stirring with a rotating movement for about 30 seconds        the capsule (3)    -   E) unscrewing the screw cap (4), and taking the platelet gel (7)        thus formed.

1. A process for preparing a platelet gel comprising mixing a plateletconcentrate with calcium chloride and batroxobin, wherein said plateletconcentrate is obtained with a process comprising: i) centrifuging 40-50ml of venous blood at 180 g for 20 minutes thereby obtaining two phasesa dark one constituted by precipitated red and white cells at the bottomof the centrifuge probe, and a clear one visible in the upper part ofthe same tube, consisting of platelet rich plasma; ii) centrifuging therecovered platelet rich plasma at 580 g for 20 minutes thereby obtainingplatelets sedimentation as “pellets” whereas the liquid supernatantconsists of platelet poor plasma; iii) suspending said pellets in analiquot of said platelet poor plasma necessary to obtain a plateletconcentrate of about 6 ml.
 2. A process for preparing a platelet gelcomprising mixing a platelet concentrate with an organic or inorganiccalcium salt and batroxobin, wherein said platelet concentrate isobtained with a process comprising: i) centrifuging 40-50 ml of venousblood at 180 g for 20 minutes thereby obtaining two phases a dark oneconstituted by precipitated red and white cells at the bottom of thecentrifuge probe, and a clear one visible in the upper part of the sametube, consisting of platelet rich plasma; ii) centrifuging the recoveredplatelet rich plasma at 580 g for 20 minutes thereby obtaining plateletssedimentation as “pellets” whereas the liquid supernatant consists ofplatelet poor plasma; iii) suspending said pellets in an aliquot of saidplatelet poor plasma necessary to obtain a platelet concentrate of about6 ml.
 3. The process according to claim 2 wherein said organic salt iscalcium gluconate.
 4. The process according to claim 2 wherein thecalcium salt is in the form of an aqueous solution containing it in anamount ranging from 5.10⁻⁵ to 0.5 M.
 5. The process according to claim 4wherein 8.10⁻⁵ M (80 μM) calcium chloride is utilised.
 6. The processaccording to claim 4, wherein 0.23 M calcium gluconate is used.
 7. Theprocess according to claims 2, wherein batroxobin has 1 thrombininternational unit/ml.
 8. A process for preparing a platelet gelaccording to claim 2 carried out with a kit comprising: a) a monousesterile capsule (3) of transparent plastic or glass material, fittedwith a screw cap (4) constituted in the upper part by a perforablemembrane (5) coated with a suitable sterile coating removable at themoment of use, said perforable membrane (5) allowing the insertion ofthe needle of a syringe (6) containing said platelet concentrate, andcontemporaneously avoiding the air contact with the inside of saidcapsule (3); b) two plug injectors (1) and (2) surmounting saidperforable membrane (5) containing respectively a calcium salt andbatroxobin; said process comprising the following steps: with a screwcap (4) constituted in the upper part by a perforable membrane (5)coated with a suitable sterile coating removable at the moment of use,said perforable membrane (5) allowing the insertion of the needle of asyringe (6) containing said platelet concentrate, and contemporaneouslyavoiding the air contact with the inside of said capsule (3); A)charging a 10 ml syringe (6) with a platelet concentrate amount rangingfrom 3 to 9 ml; B) removing the sterile coating from the perforablemembrane (5) and introducing the needle of the syringe (6) into thecapsule (3) through the perforable membrane (5); C) pressing in rapidsuccession the cap injectors (1) and (2) containing respectively 0.23 Mof calcium gluconate in 0.3-0.5 ml of batroxobin having a concentrationof 1 thrombin international unit/ml and the piston of the syringe (6)inside the capsule (3); D) gently stirring with a rotating movement forabout 30 seconds the capsule (3); E) unscrewing the screw cap (4), andtaking the platelet gel (7) thus formed, wherein the starting plateletconcentrate is obtained with a process comprising: i) centrifuging 40-50ml of venous blood at 180 g for 20 minutes thereby obtaining two phasesa dark one constituted by precipitated red and white cells at the bottomof the centrifuge probe, and a clear one visible in the upper part ofthe same tube, consisting of platelet rich plasma; ii) centrifuging therecovered platelet rich plasma at 580 g for 20 minutes thereby obtainingplatelets sedimentation as “pellets” whereas the liquid supernatantconsists of platelet poor plasma; iii) suspending said pellets in analiquot of said platelet poor plasma necessary to obtain a plateletconcentrate of about 6 ml.